Utilization of nicking properties of CRISPR-Cas12a effector for genome editing

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

have developed a Cas12a (R1226A)-based gene editing method that can act safely with reduced off-target activity in human-derived cell line.It also complements the shortcomings of wild-type Cas9 or Cas12a modules that directly induce double-strand breaks (DSBs), and has the potential to drive precise genome editing in a variety of organisms in the future.
The CRISPR-Cas12a system consists of a single CRISPR RNA (crRNA) and Cas12a endonuclease that induces target DNA recognition 15,16 .The Cas12a effector recognizes a T-rich protospacer adjacent motif (PAM) among target sequences (Fig. 1a, left inset), crRNA and target DNA hybridization induces an R-loop, and finally the active site of the RuvC domain (Fig. 1a, right inset) induces DNA cleavage 17 .The recently reported enhanced AsCas12a (en-AsCas12a) system shows that engineered amino acids near the PAM sequence (TTTN) dramatically enhances target DNA recognition, thereby increasing genome editing efficiency 18 .We optimized a nickase effector based on the en-AsCas12a (E174R, S542R, K548R) system, hereafter en-AsCas12a (R1226A), to effectively induce target-specific gene mutations with single-or dual mode of DNA targeting.To confirm the nicking property at the in-vitro level, a cleavage assay was performed by targeting the plasmid containing the target (EMX1, CCR5) nucleotide sequence (Fig. 1b,c, Supplementary Supplementary Fig. 1a, Tables 1, 2).As previously reported 11,12 , the wild-type (WT) en-AsCas12a effector showed a minor nicking property while inducing DNA double strand break (Fig. 1c, Supplementary Fig. 1b).On the other hand, the en-AsCas12a (R1226A) form showed a typical activity as a DNA nickase by mainly inducing the open circular form of the plasmid (Fig. 1c).After confirming the nicking property of the en-Cas12a (R1226A) variant, we tried to determine whether en-Cas12a (R1226A) cleaves the target-strand or non-target strand on the target DNA.To this end, we co-treated the Cas12a (R1226A) variant with SpCas9 (D10A) or SpCas9 (H840A) nickases, whose cleavage points have already been identified, and the cleaved strand of target DNA by Cas12a (R1226A) variant was identified by the formation of double strand breaks (Fig. 1d).As a result, we found that when Cas12a (R1226A) variant, SpCas9 (D10A), and SpCas9 (H840A) nickases were simultaneously treated (Fig. 1e), double strand breaks were mainly formed in case of the Cas12a (R1226A) variant and SpCas9 (D10A) nickase were co-treated (Fig. 1f).Furthermore, we analyzed the product after cleavage using the Cas12a (R1226A) variant and compared it to Cas9 (D10A or H840A) and found that a cleavage was formed at a distance of 15-18 bp from the PAM sequence (TTTN), depending on the target sequence site (Supplementary Fig. 1c).Through these results, it can be seen that, like the wild-type Cas12a nickase, the en-Cas12a (R1226A) variant also mainly cleaves the non-target strand and induces nick formation on the target DNA (Supplementary Fig. 1d).

Optimization of the distance and direction between dual Cas12a (R1226A) modules to effectively induce mutations in target DNA
Based on these cleavage test results, target-specific mutations were induced on the target gene in a humanderived cell line (HeLa) using the dual-nickase-type AsCas12a (R1226A) or SpCas9 (D10A) effector (Fig. 2, Supplementary Fig. 2).Since en-AsCas12a, an improved form of wt-AsCas12a, has shown overall superior gene editing properties in human-derived cell lines in previous studies 18 , all subsequent experiments testing Cas12a nickase-related activity were based on the en-AsCas12a effector.To confirm the directionality issue of the CRISPR nickases, the mutation induction efficiency (%) was compared for the different combination according to the directionality of each nickase (Fig. 2, Supplementary Fig. 2a,b).For each combination of SpCas9 (D10A) and en-AsCas12a (R1226A) nickase, the indel frequency (%) in the target genes (EMX1, CCR5), which is induced in a PAM-out (Fig. 2a,c,e, Supplementary Fig. 2c) and PAM-in (Fig. 2b,d,f, Supplementary Fig. 2c) fashion, was analyzed by targeted amplicon sequencing (Supplementary Figs. 3, 6, Table 3).Due to the limited issue of PAM (TTTN) recognition when applying en-AsCas12a (R1226A)-based dual nickase at each gene site, we compared the gene editing efficiency at different locations.Consequently, the indel frequency (%) induced in the direction of the PAM-out fashion (mean 12.4% for EMX1 and 5.8% for CCR5), (Fig. 2a,c,e) was higher than that in the PAM-in fashion (mean 0.9% for EMX1 and 1.0% for CCR5), (Fig. 2b,d,f) for all nickase combinations.Particularly, as the distance between the PAMs of nickase combinations increased, the overall indel frequency (%) decreased; a significant difference was observed depending on the nucleotide sequence in the target gene (EMX1, CCR5) and orientation between nickases (Fig. 2, Supplementary Fig. 2c).Most of the induced mutation pattern shows large deletions, which is possibly generated by tandem nicking of sequential nickase binding (Supplementary Figs. 3, 6).

Target-specific genome editing with decreased off-target activity using the dual-en-AsCas12a (R1226A) variant
To determine whether unintended mutations are induced (i.e., off-target effects) by the dual-and single-en-Cas12a (R1226A) variants, on-/off-target indel ratio (%) was investigated for five target loci (DNMT1, POLQ, SIRPa, AAVS1, and CCR5) (Fig. 4, Supplementary Figs. 5, 7).First, off-target candidates with up to 3 mismatched sequences compare to those of each target gene were selected based on an in-silico analysis 19 , and targeted amplicons were prepared for each predicted off-target site (Fig. 4a-e, left).Then, the mutation frequency at each offtarget site (%) was investigated by targeted amplicon sequencing (see Methods section, Supplementary Table 3).The indel mutations generated by dual en-Cas12a (R1226A) variants for each on-target and predicted off-target sites were compared with that of the wild-type en-Cas12a effector, and histograms were obtained (Fig. 4a-e, right).In a comparison of five target loci, the dual en-Cas12a (R1226A) nickase showed an editing efficiency of 27.7%, on average, compared with estimates of 64.2% for the wild-type en-Cas12a effector (WT).In comparison, for the selected off-target site, indel frequencies of 0.26% was induced by dual en-Cas12a (R1226A) variants, compared to a frequency of 9.14% for the wild-type en-Cas12a effector (WT) (Fig. 4a-e, right).

Discussion
The structures of the target DNA and guide RNA-Cas12a complex revealed to date are similar to Cas9 in that they operate in a bi-lobed form 15,20,21 .However, the cleavage domains (RuvC), which cut the target sequence after PAM recognition or R-loop formation, are located in the target DNA in different forms.Based on these structures, the Cas12a effector has a fundamentally different target DNA cleavage mechanism from that of Cas9.Recently, a process for cleaving a target nucleotide sequence was proposed based on the structure of Cas12a and biochemical experiments 22,23 .Unlike Cas9, the Cas12a complex uses a single cleavage domain (RuvC) and is thought to induce double-strand breaks with successive cleavage of DNA strands.Among the variants obtained by the introduction of mutations in the cleavage domain (RuvC), the R1226A form nicks the target DNA 15 .Our in-vitro cleavage study reproduced the result that the Cas12a (R1226A) variant can induce non-target strand cleavage, and it was hypothesized that it forms a nick due to the difficulty in accessing the target strand after the cleavage of non-target strand by RuvC domain 22 .Consistent with this cleavage mechanism, the Cas12a (R1226A) variant that induces nicking to the target DNA remains weakly capable of inducing double-strand cleavage and is weakly identified as a background form in the results (Fig. 1f).Due to these mutations, the Cas12a (R1226A) variant has a very low DNA targeting efficiency (Supplementary Fig. 8), so in this study, we used the enhanced Cas12a (R1226A) variant to effectively induce mutation of target DNA in human-derived cell lines, although concentration-saturated in-vitro cleavage experiments show similar effects (Fig. 1c).Similar to previous results for Cas9-based nickases (D10A, H840A) 5 , two Cas12a (R1226A) nickases were bound in tandem to induce an effect similar to DNA repair following double-strand break in the target DNA.Gene mutations were effectively induced (20.7% for AAVS1, 48.2% for CCR5, and 24% for DNMT1).There was a significant difference in genome editing according to the directionality and distance between the dual Cas12a (R1226A) variants (PAM out: 6.3%, PAM in: 1.4%), similar to the experimental results for a Cas9-based nickase (D10A).To effectively induce indels on the target DNA, the directionality and distance between nickases were optimized.The Cas12a (R1226A) variantbased editing system had a few key advances.By using the dual Cas12a (R1226A) variant or a combination of the established Cas9 and dual Cas12a (R1226A) variant, it was possible to accurately induce indels by simultaneously nicking the T-rich target sequence, which was difficult to target with Cas9 alone.The previously established nickase tool has been extended in terms of PAM recognition, and Cas12a nickase variants with extended PAM recognition have the potential to further expand the targeting range of gene editing (Supplementary Fig. 9) 18,24 .Notably, when a single Cas12a (R1226A) nickase was used, a target-specific mutation was induced.Hence, first, the Cas12a (R1226A) variant, which operates as a single cleavage domain, has the potential to induce unintended double-strand breaks with a low probability.Second, as described in a recent paper, indels are also induced by nicks intentionally generated on the target by the Cas12a (R1226A) variant when multiple nicks occur during DNA replication or transcription, which can mimics double-strand break-triggered DNA repair 25 .Using this single Cas12a (R1226A) variant, it is thought that precise genome editing is possible by exogenously delivered donor-based homologous recombination or inter-homolog repair in the future 25,26 .The results of this study also demonstrated that the dual en-Cas12a (R1226A) variant has the potential to decrease the unintended off-target editing compared to that of wt-enCas12a.Although the average on-target mutagenesis efficiency was lower than that of wt-en-Cas12a, we significantly reduced off-target effects by applying the principle that indels can be induced only by the simultaneous induction of dual nicks near the off-target sequence.This shows that the Cas12a-based dual nickase system can be an another effective method to overcome the off-target effects caused by the Cas12a system itself, as it is less sensitive to mismatch formation between crRNA and target DNA at PAM distal region 27 .It is thought that this principle can be applied to other recently reported type V CRISPR-Cas systems able to induce target DNA mutations 28 .
Herein, we proposed a method for target gene editing by single or dual nicking based on the CRISPR-Cas12a system.Our results demonstrate the possibility of safely inducing mutations by single nick generation in vivo, www.nature.com/scientificreports/unlike correction using homologous directed repair and non-homologous end joining after inducing DNA double-strand breaks.Furthermore, it provides a basis for replacing or supplementing technologies derived from the previous Guanine-rich PAM recognition-based CRISPR-Cas9 nickase, such as base editing 8 or prime editing 9 .This study is expected to contribute to the induction of safe and efficient gene editing in various biological systems by complementing the limitations of existing Cas9 nickase-based approaches.

H840A) proteins
For purification of en-AsCas12a (E174R, S542R, K548R), en-AsCas12a nickase (E174R, S542R, K548R, R1226A), and SpCas9 nickase (D10A) recombinant proteins, pET28a bacterial expression vectors encoding each protein were transformed into E. coli BL21(DE3) system.Transformed bacterial colonies were induced with isopropylthio-β-galactoside after culturing them in a 500 ml flask at 37 °C to OD = 0.6.After 48 h, the cultured bacteria were precipitated by centrifugation at 10,000xg at 4 °C; the culture medium was removed to obtain a bacterial pellet.Then, the pellets were treated with lysis buffer [20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM PMSF, 10 mM β-mercaptoethanol, 1% TritonX-100] to obtain intracellular proteins and dissolving the bacterial cell membrane.To effectively remove the bacterial cell membrane, lysates were subjected to sonication twice in ice water for 3 min (Qsonica, model Q700, Amplitude: 30, Process time: 3 min) and then centrifuged for 10 min at 5,000 rpm (4 °C).Nitrilotriacetic acid (Ni-NTA) resin was pretreated with wash buffer [300 mM NaCl, 20 mM Tris-HCl (pH 8.0)] to purify the (6x) His-tag-linked target recombinant protein using an affinity column.The pellet of the centrifuged solution was discarded, and the remaining supernatant and the pretreated resin were reacted at 4 °C for 90 min.To remove non-specific binding components from the cell lysate solution mixed with Ni-NTA resin, washing was performed using a wash buffer ten times the volume of the lysate.Finally, to elute the target recombinant protein, the protein was separated from the Ni-NTA resin using an elution buffer [20 mM Tris-HCl (pH 8.0), 200 mM imidazole, 300 nM NaCl]; the eluted protein solution was concentrated (Amicon Ultra) and storage buffer [200 mM NaCl, 40% glycerol, 50 mM HEPES (pH 7.5), 1 mM DTT], aliquoted and stored at -80 °C.

In-vitro transcription for guide RNA synthesis
To prepare crRNA and single-guide RNA (sgRNA) using in-vitro transcription (Supplementary Tables 1 and 2), sense and antisense DNA oligos, including target sequences for each guide RNA, were purchased from Macrogen.Sense and antisense DNA oligos were annealed under specific PCR conditions.After mixing the annealed DNA template with T7 RNA polymerase (NEB) and the reaction mixture [50 mM MgCl 2 , 100 mM rNTP (Jena Bio, NU-1014), RNase Inhibitor Murine, DEPC, 100 mM DTT, 10 × RNA polymerase reaction buffer], the reaction was carried out at 37 °C for 8 h.After the reaction, the DNA template was completely removed by reaction with DNase at 37 °C for 1 h, and only RNA was isolated using a column (MP Biomedicals, GENECLEAN® Turbo Kit).The purified RNA was lyophilized (2,000 rpm, -55 °C, 1 h) and stored for later use.

In-vitro cleavage assay for nicking property analysis
PCR amplicons containing the target sequence were obtained from purified genomic DNA (HeLa cells) using DNA primers (PCR reaction conditions, Supplementary Table 3).Then, PCR amplicons were inserted into T-blunt vector (Solgent), transformed into DH-5α E. coli cells, purified using the Plasmid Maxi Kit (Qiagen), and used as a DNA template vector for cleavage experiments.The template vector (20 ng/μL) was incubated with purified recombinant Cas12a protein (50 ng/μL) and crRNA (75 ng/μL) complex at 37 °C for 1 h.Plasmid cleavage was confirmed by 0.8% agarose gel electrophoresis of the reaction mixture.

Targeted amplicon sequencing and data analysis
To check whether the mutation of the target nucleotide sequence in the gene (CCR5 and EMX1) was induced by the gene editing approaches, next-generation sequencing was performed.Initially, PCR was performed (denaturation: 98 °C for 30 s, primer annealing: 60 °C for 15 s, and elongation: 72 °C for 15 s for 35 cycles) using a DNA primer (Supplementary Table 3) corresponding to each gene to obtain a DNA amplicon.For barcoding each DNA amplicon, nested PCR was conducted to connect the adapter and index sequences at both ends of the amplicon (denaturation: 98 °C for 30 s, primer annealing: 62 °C for 15 s, and elongation: 72 °C for 15 s for 35 cycles).
According to the manufacturer's manual, the amplicon mixture linked to the index sequence was pretreated and loaded into a mini-SEQ analyzer (Illumina MiniSeq system, SY-420-1001) to perform targeted amplicon sequencing.The saved Fastaq file was analyzed using Cas-Analyzer 29 , a web-based analysis tool, and the mutation induction efficiency (%) of the target gene was calculated (for Supplementary Figs. 2, 3, 4, 5).In addition, each indel frequency was calculated using CRISPResso2 30 and converted into a phi-chart (Supplementary Figs. 6, 7).

In-silico analysis of CRISPR effector target coverage
For target sites with Cas9 or Cas12a PAMs, we took into account that genome editing involves cleavage of DNA near NGG or TTTN PAMs and can be targeted in a sense / anti-sense direction because DNA is double helix.Therefore, for Cas9, the probability of existance of the 5'-NGG-3' sequence within 5 bases from the target base when targeting in the sense strand direction, or the probability of existance of 5'-CCN-3' when targeting in the anti-sense strand direction was calculated, respectively.Next, the probability of the AsCas12a targeting range was calculated by PAM existance of 5'-TTTN-3' or 5'-NAAA-3' sequence being present in the target site based on the sense or anti-sense manner.The probability of a target site containing both SpCas9 and AsCas12a PAMs and the probability of a site containing neither PAM was calculated as the same way. https://doi.org/10.1038/s41598-024-53648-2